Developing microsatellite duplex PCR reactions for sterlet (Acipenser ruthenus) and their application in parentage identification

The sterlet (Acipenser ruthenus) is one of the 27 sturgeon species and is well-known for its wide distribution and small body size in comparison to other sturgeons. For assessing the population genetics and parentage identification of sterlet, ten microsatellites developed for Chinese sturgeon and cross-amplified in sterlet were tested by 40 individuals of sterlet. The ten microsatellites were developed using transcriptome sequencing of Chinese sturgeon. The expected heterozygosity (HE), observed heterozygosity (HO), Shannon-Weiner diversity indices (H′) and polymorphic information content (PIC) of the 10 microsatellites ranged from 0.466 to 0.751, from 0.438 to 0.938, from 0.66 to 1.51 and from 0.368 to 0.716, respectively. Combined exclusion probability based on the genotype of pair parent known (CE-PP), one parent known (CE-2P), and no parent known (CE-1P) of the 10 microsatellites were 99.99%, 99.96%, and 99.49%, respectively. These result showed that the 10 microsatellites should be helpful for assessing the population genetics and parentage identification of sterlet.

DNA isolation. The fin of each individuals was taken and preserved in alcohol. Genomic DNA was extracted from fin of all samples of sterlet. The standard proteinase-K digestion and phenol/chloroform method was used for DNA isolation. The fin of sterlet was put in the tube with 10 µl proteinase-K for 50 min at 56 °C. Subsequently, 600 µl of extraction buffer (0.5 M Tris pH 8.0, 0.5 M EDTA, 5 M NaCl, 10% SDS) was added to this tube, and incubated for 10 min at 95 °C. Then, 500 µl phenol was put in the tube. The mixture was violented shock for 10 s and centrifuged for 15 min at 12000 g at 4 °C. 200 µl supernatant of mixture was transferred to a fresh tube. 250 µl chloroform was put this tube, and the mixture was centrifuged for 15 min at 12,000 g at 4 °C. The supernatant of mixture was transferred to another a fresh tube. 500 µl isopropanol was put this tube, and the mixture was centrifuged for 15 min at 12,000 g at 4 °C. The supernatant of mixture was discarded and 1 ml 75% ethanol was put in this tube to precipitate the pellet. And the mixture was centrifuged for 15 min at 12,000 g at 4 °C, and the supernatant of mixture was discarded. At last, the pellet was dissolved using 200 µl sterile water. 1% agarose gel was used to test DNA integrity.
Transcriptome assembly. The fin of Chinese sturgeon was used to extract RNA using Trizol (Invitrogen, USA) following the manufacturer's instructions. 1% agarose gel was used to test RNA integrity. Then, the RNA of Chinese sturgeon was used to construct cDNA libraries by using Illumina truSeq stranded mRNA sample preparation protocol. The Illumina HiSeq™ 4000 with 100 bp paired-end sequencing was used to sequence the cDNA libraries with 100 or 2 × 90 bp paired-end sequencing. SOAPnuke (v1.5.6) 14 was used to filter the raw reads of libraries according to three criteria: (i) reads with ≥ 20% bases Q ≤ 20 were removed, (ii) reads with a high proportion of unknown sequences were removed (> 5%), (iii) reads with contained adaptors (adaptors with ≤ 20% mismatches were allowed) were discarded 15 . The Trinity program (version: release-2013-08-14) was used to assemble the clean reads. The TGICL 16 was used to cluster the transcripts. The redundant sequences were eliminated and the non-redundant sequences of > 200 bp were retained.
Identification of microsatellites. The Microsatellite Identification tool 17 was used to select sequences that is constraining perfect repeat motifs of 4 bp from the libraries data. And the Microsatellite Identification tool was set for search criteria for identification of at least 6 repeat units. The Primer Premier 5.0 software 18 was used to design microsatellite primers from all the selected sequences. The primer pairs flanking each microsatellite was identified with a annealing temperature of an optimum at 60 °C, PCR products with expected lengths between 100 and 400 bp.
Marker selection. The DNA of 16 randomly individuals of sterlet were used to screen for polymorphic microsatellites and to optimize amplification conditions. PCR amplification was performed in 25 μl volumes containing 0.25 μM each primer, 1.5 mM MgCl 2 , 0.25 U of Taq polymerase (Takara, China), 0.25 μM PCR buffer (Takara, China), 0.25 μM dNTPs, about 50-100 ng of template DNA, and ultra-pure water. PCR amplification was conducted under the following conditions: an initial step at 94 °C for 3 min, followed by 35 cycles of denaturing 30 s at 94ºC, 60 °C for 30 s, and 72 °C for 30 s, with a final prolonging at 72 °C for 10 min. The PCR products were resolved with 10% polyacrylamide gel electrophoresis (PAGE). The pBR322 DNA/Mspl marker (Takara) was used as a molecular size standard.

Establishing duplex PCR panels for sterlet and their application in parentage identification.
According to the result of marker selection of sterlet, duplex PCR reactions was chosen. The primer dimers and potential hairpin structures must be avoided in choosing of duplex PCR reactions. If primer dimers and potential hairpin structures appear in PCR reaction products, this combination of primers is filtered out. Five duplex PCR reactions were assembled, each containing two microsatellites. The duplex PCR reactions were performed at a total volume of 25 μl, containing 0.25 μM each primer, 1.5 mM MgCl 2 , 0.5 U of Taq polymerase (Takara, China), 0.5 μM PCR buffer (Takara, China), 0.5 μM dNTPs, about 80-100 ng of template DNA, and ultra-pure water. The duplex PCR reactions were performed under the following profile: an initial step at 94 °C for 3 min, followed by 35 cycles of denaturing 30 s at 94 °C, 60 °C for 30 s, and 72 °C for 30 s, with a final prolonging at 72ºC for 10 min. The PCR products were resolved with 10% polyacrylamide gel electrophoresis (PAGE). The pBR322 DNA/Mspl marker (Takara) was used as a molecular size standard. The selected microsatellites for duplex PCR reactions were used for parentage identification of the full-sib families of sterlet.
n j=i+1 2P 2 i P 2 j , P i and P j are the frequencies of I and J allele in each microsatellite loci. The Gene Marker software was used to calculate the size of allele. Microsoft Office Excel 2007 was used to process the genotypic data. Based on the allele phenotypes, a dendrogram of ten random individuals of sterlet was constructed using MEGA software 20 . The FaMoz software 21 was used to calculate the exclusion and combined exclusion probability based on the allele frequencies. During the analysis the genetic data of the experiment, the dropout and false allele should be avoid.

Results
In this study, a total of 160 microsatellites primers were designed to develop duplex PCR reactions. 16 sterlet individuals were used to test the 160 microsatellites, and 10 microsatellites showed clearly polymorphism ( Table 1). The observed allele number of the 10 microsatellites ranged from 2 to 6. The expected heterozygosity (H E ), observed heterozygosity (H O ), Shannon-Weiner diversity indices (H′) and polymorphic information content (PIC) of the 10 microsatellites ranged from 0.466 to 0.751, from 0.438 to 0.938, from 0.66 to 1.51 and from 0.368 to 0.716, respectively (Table 2). According to the results for the 10 microsatellites tested in the 16 sterlet individuals, we build five duplex PCR reactions ( Table 1). The 16 sterlet individuals were accurately reconstructed in the dendrogram based in the 10 microsatellites by Mega software (Fig. 1). The five duplex PCR reactions were also used in the parentage analysis of the full-sib families of sterlet. Combined exclusion probability based on the genotype of pair parent known (CE-PP), one parent known (CE-2P), and no parent known (CE-1P) increased with the number of microsatellites (Fig. 2). Combined exclusion probabilities based on the genotype of CE-PP, CE-2P, and CE-1P of the 10 microsatellites were 99.99%, 99.96%, and 99.49%, respectively. Combined exclusion probability of CE-PP, CE-2P, and CE-1P can be reach 99% when used 10, 8, 5 microsatellites, respectively. These result showed that the 10 microsatellites should be helpful for parentage identification of sterlet.  www.nature.com/scientificreports/

Discussion
Application of microsatellite markers play important role in genetic conservation and management of fish, and many studies have demonstrated that microsatellite is usefulness for the broodstock management and assessment of population structures [22][23][24][25][26] . In the present study, five duplex PCR reactions were built to be used in the population genetics and parentage identification for sterlet. The microsatellite for sterlet have been developed in www.nature.com/scientificreports/ previous studies 12,27 . However, only seven tetranucleotide microsatellites were reported 12 . The risk of obtaining false alleles can be reduced by using tetranucleotide microsatellites instead of dinucleotide microsatellites 28 . The dinucleotide microsatellites are more likely to occur as shadow bands than tetranucleotide microsatellites during the PCR process 29 . Furthermore, tetranucleotide microsatellites are more stable and accurate than dinucleotide microsatellites 30 . Therefore, the 10 tetranucleotide microsatellites should be helpful for genetic study of sterlet. The observed heterozygosity and expected heterozygosity of the 10 microsatellites in this study is higher than the previous microsatellites 12,27 . In this study, 9 of 10 microsatellites with PIC > 0.5, which is higher than the microsatellites that be reported in beluga sturgeon 31 , Amur sturgeon 10 , however, is lower than the microsatellites that be reported in Chinese sturgeon 6 , Dabry's Sturgeon 9 . Although the method of alleles scoring (PAGE) used in this study is cheap and quick, it has many drawbacks, such as result interpretation error, misreading false allele and so on. In this study, there is no evidence of stutter bands, null alleles, or large allele dropout. The reason for this result may be due to the small sample size. These result suggested that the 10 microsatellites were helpful in studying of the population genetics of sterlet.
The previous study have showed that the CE-1P and CE-2P for parentage identification of Nile tilapia were 0.967 and 0.9999 by using 7 microsatellites 32 . Using four groups of fluorescent-labeled multiple capillary for parentage assignment, the assignment success rate of O. niloticus, O. niloticus × O. aureus, O. aureus, and their mixed population reached 100% when 7, 8, 9, and 12 microsatellites, respectively 33 . The parentage identification success rate was highly correlated with the allelic polymorphism of microsatellite 34 . The 10 microsatellites in this study have showed high allelic polymorphism, suggesting that they are suitable for parentage identification of sterlet. They can be useful tools to characterize the structure and genetic diversity of sterlet, to appropriate breeding schemes and establish broodstocks for supportive stocking programs, to aid the development of conservation measures as well as to monitor genetic changes in farmed strains. Although the 10 microsatellites presented here was meaning for sterlet parentage identification, this methodology still has some limitations: (a) The PIC values of the 10 microsatellites in this study are not sufficiently high to reduce the number of microsatellites for parentage assignment; (b) More stable microsatellites need to be further screened.
In conclusion, these five duplex PCR reactions, which consist of 10 microsatellites, should be helpful for assessing the population genetics and parentage identification of sterlet.

Data availability
Data are available from the corresponding author upon reasonable request.